Dna enzymes

ABSTRACT

Methods for the selection of novel signaling allosteric DNA enzymes are provided. In particular, fluorescent signaling allosteric DNA enzymes are described. The selection system is based on the cleavage of an ribonucleotide flanked by a fluorophore modified nucleotide and a quencher modified oligonucleotide. Both cis-acting and trans-acting allosteric DNA enzymes are identified, as well as aptamer/DNA enzyme conjugates.

FIELD OF THE INVENTION

The present invention is directed to methods for the detection andisolation of nucleic acid enzymes which possess desired characteristics.It is also directed to the enzymes isolated according to the methodsdescribed and assays based on the use of those enzymes. In particular,it relates to the generation of fluorescent signaling reporters withsubstrate and/or reaction specificity.

BACKGROUND OF THE INVENTION

Throughout this application, various references are cited in parenthesesto describe more fully the state of the art to which this inventionpertains. The disclosures of these references are hereby incorporated byreference into the present disclosure, and for convenience thereferences are listed in the list of references appended hereto.

Over the past decade, there have been significant advances in thedevelopment of selective biosensors based on the use of DNA as abiorecognition element. While the majority of DNA based sensors aredesigned to detect complementary DNA, many recent reports havedemonstrated that single-stranded DNA can also form intricate tertiarystructures that allow it to selectively bind to non-DNA targets (socalled aptamers)^(1,2) or perform catalysis of chemical reactions.^(3,4)To date, over 100 DNA sequences have been reported for facilitating manytypes of chemical transformations.⁵ In spite of having very limitedchemical functionalities, deoxyribozymes that perform catalysis withsurprising efficiency have been reported in a number of studies.⁶ Forexample, a small DNA enzyme known as 10-23 performs site-specific RNAcleavage with a very impressive k_(cat) of ˜10 min⁻¹.⁷ It is clear thatthe lack of a 2_-hydroxyl group in DNA relative to RNA is not adetriment to catalytic performance. Furthermore, the catalyticcapabilities of DNA can be enhanced through the use of metal ions⁸ andsmall-molecule cofactors⁹ as well as through modification with chemicalfunctionalities that are useful for catalysis.¹⁰ Furthermore, whencompared to ribozymes, deoxyribozymes are easier to prepare and moreresistant to chemical and enzymatic degradation, and therefore, properlyengineered and catalytically efficient DNA enzymes are very desirableelements for the construction of rugged biosensors.

Allosteric ribozymes and deoxyribozymes have tremendous potential forwide-ranging applications in the diagnostic, biosensing and drugscreening fields. The use of deoxyribozymes with fast catalytic ratesand large turnover numbers allows for the engineering of effectiveallosteric DNA enzymes for practical applications where rapid enzymaticaction is essential. To engineer catalytic DNA probes for detectiondirected applications, it is very desirable to use DNA enzymes that cancouple enzymatic activity with fluorescence signaling capability so thateasy and fast detection can be performed in real time without the needfor time-consuming separation steps.

SUMMARY OF THE INVENTION

The present invention provides a de novo fluorescence-generatingRNA-cleaving DNA enzyme system that maintains low backgroundfluorescence yet is capable of generating a very large fluorescentsignal upon RNA cleavage, and which exhibits a very large catalytic rateconstant. A method for the detection and isolation of DNA enzymes isprovided. The RNA-cleaving DNA enzyme of the present invention uniquelylink chemical catalysis with real-time fluorescence signalingcapability. Two specific examples of this system, a cis-acting enzymecapable of autocatalysis, and a trans-acting enzyme that acts on aspecific chimeric substrate, are provided. Development of an allostericDNA enzyme controlled by aptamer target binding is also demonstrated. Ina preferred embodiment, a known ATP aptamer is conjugated to thecis-acting enzyme.

In one aspect of the invention, there is provided a signaling DNA enzymeconstruct. The construct comprises a) an enzymatic DNA sequence capableof cleaving at a ribonucleotide site and b) a DNA chain having aribonucleotide linkage flaked by a fluorophore modified obigonucleotideand a quencher modified obigonucleotide in sufficient proximity to eachother whereby, in the absence of catalysis, fluorescence from thefluorophore is quenched by the quencher.

In a preferred embodiment, the enzymatic DNA sequence is a cis-actingenzyme having the sequence defined in SEQ.ID.NO.7 or SEQ. ID. NO.: 8.

In another preferred embodiment, the enzymatic DNA sequence is atrans-acting DNA enzyme having the sequence of SEQ.ID.NO. 9.

In a further aspect of the invention, a signaling DNA enzyme constructcomprises an aptamer sequence conjugated to the enzymatic DNA sequence.

In a preferred embodiment, the signaling DNA enzyme/aptamer constructcomprises the sequence of SEQ.ID.NO. 10.

In another aspect of the invention, there is provided a method ofselecting an RNA-cleaving catalytic DNA molecule. The method comprisesthe following steps:

-   1. synthesizing a single-stranded DNA molecule having a    ribonucleotide flanked by a fluorophore labeled nucleotide on one    side and a quencher modified nucleotide on the other side, and    having a random sequence insertion site;-   2. inserting random DNA sequences into the insertion site to provide    a candidate DNA molecule;-   3. incubating the candidate DNA molecule in the presence of a    co-factor; and-   4. detecting the presence or absence of a fluorescent signal,    wherein the signal is generated when cleavage occurs at the    ribonucleotide thereby separating the quencher from the fluorophore.

The present invention also provides another method for the selection ofan enzymatic DNA sequence. The method comprises the steps of:

-   -   providing a library of oligonucleotides comprising random        sequences;    -   ligating the oligonucleotides to an acceptor sequence comprising        a ribonucleotide linkage flanked by a fluorophore modified        nucleotide and a quencher modified oligonucleotide;    -   determining whether a fluorescent signal is generated due to        cleavage of the ribonucleotide linkage; and    -   amplifying sequences which were cleaved at the ribonucleotide.

In a further aspect of the invention, there is provided a method forselecting autocatalytic DNA from a random pool of DNA, said methodcomprising the steps of:

-   1. providing a pool of single stranded DNA molecules comprising a    first predetermined sequence, a random sequence and a second    predetermined sequence;-   2. ligating said single stranded DNA molecules to an acceptor DNA    molecule comprising at least one ribonucleotide flanked by a    fluorophore modified oligonulceotide and a quencher modified    nucleotide at a ligation junction;-   3. isolating a single stranded ligated oligonucleotide;-   4. incubating said ligated oligonucleotide in the presence of    cofactors;-   5. measuring RNA-cleavage activity by PAGE;-   6. isolating DNA molecules which had been cleaved at a    ribonucleotide.

In a preferred embodiment, the DNA selected by the above describedmethod is subjected to further rounds of selection. This comprises thesteps of:

-   7. a first PCR amplification using a first primer which is    complementary to a region of the ligated DNA encompassing the    ligation junction and a second primer which is complementary to the    second predetermined region;-   8. a second PCR amplification using a ribo-terminated third primer    to provide a double stranded DNA product having a ribonucleotide at    the ligation junction;-   9. cleavage of the double stranded DNA product at the    ribonucleotide;-   10. isolation of single stranded DNA molecules as defined in step 1;    and-   11. a repeat of steps ii) to x) until a sufficient degree of    selection is achieved.

The present invention also provides a method for the selection of anaptamer sequence specific for a desired target. The method comprisesconjugating random sequences to a signaling autocatalytic DNA enzyme,incubating the conjugated sequence in the presence of the desired targetand determining the fluorescent intensity of the solution. In apreferred embodiment, an assay for the detection of important biologicaltargets is provided.

The present invention also provides a kit for the selection of anenzymatic DNA sequence. In one preferred embodiment the kit comprises aDNA construct comprising a DNA claim with a ribonucleotide linkageflanked by a fluorophore modified nucleotide and a quencher modifiedoligonucleotide and a sequence adapted for insertion of randomoligonucleotides. In another embodiment, kit includes a library DNAadapted for insertion of random or known sequences, an acceptor DNAcomprising a ribonucleotide flanked by a fluorophore modified nucleotideand a quencher modified oligonucleotide and primers for PCRamplification of RNA cleaving sequences.

In yet another aspect, a method for the detection of a required factoris provided. The method comprises providing a signaling DNA construct,introducing a sample; and determining whether a signal is generated. Ina preferred embodiment a method for the detection of metal ions or smallmolecules is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention are described below withreference to the drawings, wherein:

FIG. 1 is a schematic representation of the signal generated uponcleavage of a DNA chain at a ribonucleotide linkage;

FIG. 2 illustrates the selection of a signaling sequence;

FIG. 3A is a schematic representation of a method for the selection ofan RNA-cleaving DNA enzyme;

FIG. 3B illustrates exemplary sequences used to select a DNA enzymeaccording to the method illustrated in FIG. 3A;

FIG. 3C illustrates graphically the enzymatic activity under variousselection conditions;

FIG. 4A illustrates the sequence of a cis-acting DNA enzyme;

FIG. 4B illustrates the signaling properties of the enzyme illustratedin FIG. 4A;

FIG. 5 illustrates the effect of 3=truncations on signaling activity;

FIG. 6A illustrates the secondary structure of the cis-acting DNAenzyme, DEC22-18A;

FIG. 6B illustrates the proposed secondary structure of a trans-actingDNA enzyme, DET22-18;

FIG. 6C is a graphical representation of the kinetic analysis ofDET22-18;

FIG. 7A demonstrates the real-time signaling capability of DET22-18 whenthe enzyme is in excess;

FIG. 7B demonstrates the real-time signaling capability of DET22-18 whenthe substrate is in excess;

FIG. 8A illustrates an allosteric DNAS enzyme where an aptamer sequenceis coupled to a signaling DNA enzyme;

FIG. 8B illustrates graphically an activation assay based onintroduction of the aptamer target; and

FIG. 8C demonstrates the target specificity of the signaling construct.

DETAILED DESCRIPTION

The present invention is directed to enzymes which cleave a substrate ata defined cleavage site. In particular, DNA-containing molecules capableof functioning as enzymatic reporters and methods for their isolationare provided.

Throughout this specification the terms enzymatic DNA molecule,catalytic DNA, DNA enzyme, DNAzyme and deoxyribozyme are usedinterchangeably. Enzymatically active portions are also encompassedwithin the terms. The enzymatic DNA molecules of the present inventionmay be modified by mutations, deletions and/or additions and they maycomprise nucleotide analogs. The enzymatic DNA molecules of the presentinvention cleave an oligonucleotide substrate. Both cis-acting andtrans-acting enzymes are encompassed.

Catalytic DNA molecules cleave phosphodiester bonds and thus have manyuses both in pharmaceutical/medical applications and in everyday life.

The present invention provides a rapid fluorescence based system for thedetection of catalytic DNA molecules that can cleave RNA. A signalingoligonucleotide is synthesized which includes a ribonucleotide. Afluorophore-modified nucleotide is located on one side (e.g. upstream)of the ribonucleotide and a quencher-modified nucleotide is located onthe other side (e.g. downstream). It is clearly apparent that theopposite orientation (i.e. the fluorophore-modified nucleotide locateddownstream of the ribonucleotide and the quencher-modified nucleotideupstream) would also be functional. The quencher-modified nucleotideshould be sufficiently close to the fluorophore-modified nucleotide toprovide a low background of fluorescence. The signaling oligonucleotideis coupled to random sequences. If the random sequence comprises a DNAenzyme capable of cleaving the signaling oligonucleotide at theribonucleotide, the fluorophore and the quencher become separated and asignificant increase in the fluorescent signal can be detected.

The present invention allows for the selection and isolation of a DNAenzyme based on the generation of fluorescent signal. In one aspect ofthe invention, a signaling DNA enzyme reporter system based on RNAcleavage is provided. The general concept is illustrated in FIG. 1. Areporter 10 is provided which comprises a DNA chain 12 having an RNAlinkage 14 embedded therein. A fluorophore 16 is linked to the chain 12on one side of the RNA linkage and a quencher 18 is linked to the chainon the other side of the RNA linkage. The fluorophore and the quencherare sufficiently close to each other to provide efficient quenching ofthe fluorescence from the fluorophore by the quencher. This alsominimizes false positives. When the RNA linkage is cleaved, thefluorophore and the quencher separate and a fluorescent signal isgenerated. This system can be used to detect the presence of any moietythat cleaves RNA. This system can also be used to detect the presence ofco-factors required by an RNA-cleaving enzyme.

An optimal signaling DNA reporter will have a good signal to noiseratio. There is low background in the absence of any enzymatic activityand a strong signal is generated when cleavage has occurred. The effectof the distance between the fluorophore and the quencher on theseproperties can be assessed using constructs similar to those shown inFIG. 2 and discussed further in Example 2. A series of constructs wherethe fluorophore and quencher were spaced at different distances areprepared. It is well known that RNA can be fragmented with base and NaOHis known to break down RNA and not DNA. Thus, NaOH can be added to theconstructs and the fold change in fluorescence can be determined. Inthis manner a signaling reporter based on cleavage at a particular sitecan be prepared. Generally, the fluorophore and quencher should besufficiently close to give a low background in the absence of cleavageand provide a good signal to noise ratio.

In another aspect, the present invention provides a method for theselection and isolation of fluorescent signaling RNA-cleavingautocatalytic DNA molecules. Basically, a DNA construct is providedwhich includes a ribonucleotide flanked by a fluorophore modifiedoligonucleotide and a quencher-modified oligonucleofide. The constructalso includes a site for insertion of random nucleotide sequences. Ifthe inserted sequence has RNA cleaving activity, the ribonucleotidelinkage is cleaved and the fluorophore is separated from the quencherand a fluorescent signal is generated.

Several rounds of selection are preferably done to enrich for thecatalytic sequence. In a preferred embodiment a selection scheme similarto the one shown in FIG. 3A and discussed in Example 5 is used to enrichand select the RNA cleaving DNA enzyme.

The selection scheme of the present invention comprises generating apool of single stranded DNA molecules comprising a random sequenceflanked by a predetermined 5=sequence and a predetermined 3=sequence.These DNA molecules are referred to as Alibrary≅DNA. An oligonucleotide,referred to herein as an Aacceptor≅oligonucleotide, comprises afluorophore modified nucleotide, a quencher modified nucleotide and aribonucleotide linkage positioned between the fluorophore and thequencher. Another oligonucleotide, termed Atemplate DNA≅ is alsoprovided. Template DNA comprises a first sequence which is at leastpartially complementary to the sequence of the acceptor oligonucleotideand a second sequence which is at least partially complementary to thepredetermined 5=sequence of the library DNA. Due to the complementarityof the sequences, the template DNA forms a duplex structure with theacceptor oligonucleotide and the library DNA and brings them intoproximity. When a ligase is introduced, the library DNA is ligated tothe acceptor oligonucleotide to form a ligated molecule. The duplexstructure is dissociated and the ligated molecule can be separated fromthe template DNA by PAGE.

A particular feature of present invention is that it permits selectionand isolation of an enzyme on the basis of fluorescent signaling. It isclearly apparent that the selection scheme of the present invention isnot limited to the particular sequences shown in FIG. 3. The generalscheme can be used to select a variety of DNA enzymes having differentcharacteristics.

Enzymatic DNA molecules that require the presence of co-factors such assmall molecules, peptides, metal ions, metabolites, sugars, nucleicacids, etc. are selected by incubating the ligated molecule in thepresence of that factor. If the ligated molecule comprises a DNA enzymethat is responsive to that factor, cleavage will occur at theribonucleotide linkage. This will result in the generation of afluorescent signal as the fluorophore and quencher become separated. Anexample of this is shown in step III of FIG. 3 when metal ions areintroduced.

The autocatalytic molecules can then be enriched through a series ofpolymerase chain reactions. Since the autocatalytic DNA will have thepredetermined 3=sequence of the library DNA, a primer complementary tothat sequence can be used. This primer is termed P1. A second primer,P2, comprises a sequence complementary to the acceptor oligonucleotideand the conserved 5=sequence of the pool DNA. PCR with these primerswill generate DNA molecules having the sequence of the ligated DNA withthe exception of the ribonucleotide. The ribonucleotide is thenintroduced using a third primer, P3, which is ribo-terminated. Afteramplification, the DNA is treated with an RNA cleaving moiety, such asNaOH. The cleaved DNA is subjected to PAGE purification and DNAphosphorylation. The 5=phosphorylated DNA is used to initiate a furtherround of selection. Using this strategy highly selective reporters caneven be regenerated in situ.

It is clearly apparent to one skilled in the art that the method isgenerally applicable and is not limited to the specific nucleotidesequences shown in FIG. 3.

The DNA enzyme can be initially selected and enriched by going through anumber of selection rounds. In addition, the time allowed for the selfcleavage reaction can be gradually decreased to select for the mostefficient DNA enzymes as shown in FIG. 3C and discussed further inExample 5. In a preferred embodiment, the techniques will result inclones comprising a single class of DNA enzyme after several rounds ofselection.

An RNA-cleaving DNA enzyme was isolated using the above-describedmethodology and was termed DEC22-18. The terminology is based on DNAenzyme, cis-acting, 22 rounds of selection, clone 18. DEC22-18 is alarge DNA molecule consisting of 109 nucleotides. The sequence of thisenzyme is shown in FIG. 4A and in SEQIDNO:7. The catalytic activity ofthis molecule was confirmed as illustrated in FIG. 4B and describedfurther in Example 7.

In an aspect of the invention, the minimal sequence required forcatalytic activity is determined by doing a series of nucleotidetruncations and measuring the enzymatic activity of the truncatedmolecules. In an exemplary embodiment of the present invention, DEC22-18was subjected to a series of 3=truncations. The truncation experimentsare illustrated in FIG. 5 and described more fully in Example 8. Thetruncation of the last 26 nucleotides resulted in an enzyme termedDEC18-22A which is a highly efficient enzyme. Once a primaryoligonucleotide structure is known, the secondary structure can bepredicted using various algorithms. The secondary structure of DEC22-18Awas predicted using the M-Fold program(http://bioinfo.math.rpi.edu/˜mfold/dna) and is shown in FIG. 6A.

DEC22-18A is a cis-acting enzyme. Based on the secondary structure ofDEC 22-18A, it is possible to design a trans-acting DNA enzyme system. Atrans-acting DNA enzyme, DET22-18, is also provided. The structure ofDET22-18 and illustration of its signaling properties are illustrated inFIGS. 6 and 7 and described more fully in Examples 9 and 10.

The RNA-cleaving DNA enzymes of the present invention can also be usedto design a signaling allosteric deoxyribozyme. An aptamer sequence isconjugated to a DNA enzyme having a stem-loop secondary structure. Anexemplary signaling allosteric deoxyribozyme is shown in FIG. 8 anddiscussed further in Example 11. In a preferred embodiment, a weak stemis used to conjugate the aptamer sequence to the enzyme sequence. In theabsence of the aptamer target, the stem is weak and so the catalyticactivity is weak. In the presence of the target, the formation of thestem is promoted and there is a concomitant increase in catalyticactivity due to the formation of the secondary structure. The DNA enzymemoiety is modified as described above to include a ribonucleotideflanked by a fluorophore and a quencher. In the absence of aptamertarget, the stem formation is weak and there is little or no cleavage atthe ribonucleotide. Thus, in the absence of target, the Fluorophore andquencher remain in close proximity and the fluorescence is quenched. Inthe presence of the aptamer target, the DNA enzyme assumes its secondarystructure and cleavage occurs at the ribonucleotide resulting in afluorescent signal being generated. Thus, the signaling, allosteric DNAenzyme or Aaptazyme≅ can be used to detect the presence of a targetmolecule.

It is clearly apparent that the signaling DNA enzymes of the presentinvention can be conjugated to various aptamer sequences using a varietyof techniques. Based on the ease with which cleavage can be detected bya fluorescent signal, the signaling enzymes of the present invention canbe used to identify aptamer sequences. Random sequences can beconjugated to the deoxyribozyme domain and tested for their ability tobind to various targets.

In a preferred embodiment, a signaling allosteric DNA enzyme comprisingDE22-18A conjugated to an aptamer sequence is provided. In a preferredembodiment a signaling allosteric DNA enzyme comprising DE22-18Aconjugated to an ATP binding aptamer is provided. The secondarystructure of this conjugated DNA molecule is shown in FIG. 8A and thesequence is described in SEQ.ID.NO.:10. The target reportingcapabilities of this molecules are illustrated in FIG. 8B and 8C anddiscussed further in Example 11. It is clearly apparent the ATPaptamer/DEC22-18 aptazyme detects the presence of ATP and that thesignal generated in target specific. Signaling allosteric DNA enzymesincorporating other aptamer sequences are encompassed within theinvention.

The signaling DNA enzymes of the present invention are useful in avariety of ways. The signaling DNA enzyme systems of the presentinvention are well-suited for solution-based assays for detectingspecific analytes. Such an assay is easy to use and the detection isextremely rapid since there is no need to have a separation step or toadd fluorogenic reagents. The present invention also has the advantagethat because selection is done with the fluorophore and quencher inposition, the risk of altering the activity of the catalytic DNA bypost-labeling reactions is eliminated.

The DNA molecules of the present invention can also be immobilized ontoa variety of surfaces, including quartz, glass, silica, various metalsand any polymers. The DNA can be immobilized onto optical fibers, planarwaveguides or microscope slides. The DNA can be applied as a monolayeror multilayer or it can be entrapped in a polymer solution.

Throughout this description, the use of fluorescein as the fluorophoreand DABCYL as the quencher has been described. It is clearly apparentthat alternative probe systems that have as effective or enhancedphotostability and better scatter rejection can be used. For example,very long life-time probes based on Eu(III) and Tb(III), Ru(II) probesand long-wavelength probes such as Texas Red can also be used. Inaddition, FRET acceptors and FRET donors can be used to generate ameasurable fluorescent signal. The system of the present invention isalso well suited of the construction of wave-length shifting fluorescentreporters.

The present invention also provides a kit for the selection of anenzymatic DNA sequence. In one preferred embodiment the kit comprises aDNA construct comprising a DNA claim with a ribonucleotide linkageflanked by a fluorophore modified nucleotide and a quencher modifiedoligonucleotide and a sequence adapted for insertion of randomoligonucleotides. In another embodiment, kit includes a library DNAadapted for insertion of random or known sequences, an acceptor DNAcomprising a ribonucleotide flanked by a fluorophore modified nucleotideand a quencher modified oligonucleotide and primers for PCRamplification of RNA cleaving sequences.

The present invention provides signaling allosteric DNA enzymes andmethods for their detection, selection and amplification. Both acis-acting RNA-cleaving DNA enzyme, DEC22-18, and a related trans-actingDNA enzyme, DET22-18, that have uniquely synchronized chemicalcatalysis/real-time signaling capabilities are provided. DEC22-18 has aunique structural feature wherein the enzyme and substrate are presentwithin the same molecule, leading to an autocatalytic system capable ofgenerating a large fluorescence signal with appropriate divalent metalions. An advantage of such a system is that since both the catalytic andsignaling components are present in a single molecule, Areagentless≅sensors can be developed based on immobilization of the DNAzyme onto asuitable surface such as that of an optical fiber. In this case, onlythe presence of the appropriate target would be required to generate asignal. Given the large k_(obs) value and the potential to achieve verysignificant enhancement in fluorescence intensity from this system,rapid and sensitive detection of target molecules can be achieved withsuch a reporter.

The trans-acting DNAzyme DET22-18 is a true enzyme with a k_(cat) of ˜7min⁻¹, making it one of the fastest DNA enzyme reported to date. The58-nt DNA enzyme cleaves a chimeric RNA/DNA substrate at the lone RNAlinkage surrounded by a closely spaced fluorophore-quencher pair. Thisunique structure permits the synchronization of chemical cleavage withfluorescence signaling. The extremely short distance between F and Qgives rise to the maximal fluorescence quenching in the startingsubstrate (for both cis and trans reactions) and results in a very largefluorescence enhancement upon chemical catalysis. At the same time, thecovalent integration of F and Q within the same substrate prohibitsundesirable long-range movement of the fluorophore and the quencher awayfrom each other so that the potential for false signaling that does notoriginate from chemical catalysis can be minimized. The signaling DNAenzymes of the present invention have the ability for fast chemicalaction, synchronized catalysis-signaling capability, excellentfluorescence signaling properties (low background fluorescence, largesignal enhancement, and minimal potential for false signaling), and asimple stem-loop structure. This makes them ideal DNA enzymes forengineering useful allosteric deoxyribozyme biosensors with exceptionalreal-time detection sensitivity and accuracy. A large number of similarDNA enzymes carrying different fluorophores and quenchers can be createdvery easily with the similar strategy used for the creation of DEC22-18and DET22-18. Such DNA enzymes are useful in setting up various forms ofmultiplexed assays for the detection of important biological targets.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitation.

EXAMPLES

The examples are described for the purposes of illustration and are notintended to limit the scope of the invention.

Methods of synthetic chemistry, protein and peptide chemistry andmolecular biology, referred to but not explicitly described in thisdisclosure and examples are reported in the scientific literature andare well known to those skilled in the art.

Example 1 Oligonucleotides

Standard oligonucleotides were prepared by automated DNA synthesis usingcyanoethylphosphoramidite chemistry (Keck Biotechnology ResourceLaboratory, Yale University; Central Facility, McMaster University).Random-sequence DNA libraries were synthesized using an equimolarmixture of the four standard phosphoramidites. DNA oligonucleotides werepurified by 10% preparative denaturing (8 M urea) polyacrylamide gelelectrophoresis (PAGE) and their concentrations were determinedspectroscopically and calculated using the Biopolymer Calculatorprogram. (available at http://paris.chem.yale.edu)

Fluorescein and 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL) labelswere incorporated into the DNA during automated DNA synthesis usingFluorescein-dT amidite and DABCYL-dT amidite (Glen Research, Sterling,Va). The adenine ribonucleotide linkage was also introduced duringsolid-state synthesis using A-TOM-CE Phosphoramidite (Glen Research).Fluorescein and DABCYL modified oligonucleotides were purified byreverse phase liquid chromatography (HPLC) performed on aBeckman-Coulter HPLC System Gold with a 168 Diode Array detector. TheHPLC column used was an Agilent Zorbax ODS C18 Column with dimensions of4.6 mm_(—)250 mm and a 5-micron bead diameter. Elution was achievedusing a two-buffer system with buffer A being 0.1 M triethylammoniumacetate (TEAA, pH 6.5) and buffer B being pure acetonitrile. The bestseparation results were achieved using a non-linear elution gradient (0%B for 5 min, 10% B to 30% B over 95 min) at a flow rate of 0.5 ml/min.The main peak was found to have very strong absorption at both 260 nmand 491 nm.

The TOM protective group on the 2_-hydroxyl group of the RNA linkage wasremoved by incubation with 150 _l of 1M tetrabutylammonium fluoride(TBAF) in THF at 60° C. with shaking for 6 hr, followed by the additionof 250 _l of 100 mM Tris (pH 8.3) and further incubation with shakingfor 30 min at 37° C. The DNA was recovered using ethanol precipitation,dissolved in water containing 0.01% SDS, and the tetrabutylammonium saltwas removed by centrifugation using a spin column (Nanosep 3K Omega,Pall Corp., Ann Arbor, Mich.).

Nucleoside 5_-triphosphates, [_(—) ³²P]ATP and [_(—) ³²P]dGTP werepurchased from Amersham Pharmacia. Taq DNA polymerase, T4 DNA ligase andT4 polynucleotide kinase (PNK) were purchased from MBI Fermentas. Allother chemical reagents were purchased from Sigma.

Example 2 Design of Oligonucleotides for Optimal Fluorescence-quenching

An RNA-cleavage based signaling DNA enzyme reporter that had a lowbackground fluorescence in its inactive state under any given conditionbut could generate a large fluorescence signal upon cleavage of thesingle RNA linkage embedded in a DNA chain and flanked by a covalentlylinked fluorophore and quencher pair was created. This arrangement notonly results in very efficient fluorescence-quenching because of theshort distance between the fluorophore and the quencher, but alsominimizes false positives because the quencher cannot be separated fromthe fluorophore until the RNA linkage is cleaved. To determine theoptimal distance between the fluorophore and the quencher, a series ofDNA oligonucleotides with the modifications as shown in FIG. 2A (F:fluorescein-dT; Q: DABCYL-dT; Ar: adenine ribonucleotide) weresynthesized. The cleavage-dependent signaling behavior of these DNAmolecules was assessed by treatment with 0.25M NaOH, and the data areshown in FIG. 2B, where F₀ and F are the fluorescence intensities of arelevant DNA solution measured immediately after the addition of 0.25 MNaOH (RNA cleavage yet to occur) and after an incubation for 20 hr (fullRNA cleavage¹⁹). In this example, F1QDNA had the most significantfluorescence change (with an increase in intensity of ˜70-fold),followed by F2QDNA (˜30-fold). F3DNA produced a fluorescence enhancementof around 4-fold. The decrease in fluorescence enhancement with distanceresulted from a higher value for F₀ as distance increased, owing to lessefficient quenching. All FxQDNA systems (x=1-3) reached final intensityvalues that were similar to FDNA.

Example 3 Fluorescence Measurements

All measurements were made with 400 μl solutions on a Cary EclipseFluorescence Spectrophotometer (Varian). The excitation was set at 490nm and emission at 520 nm.

Example 4 Kinetic Analyses

A typical reaction involved the following steps: (1) heat denaturationof DNA in water for 30 sec at 90 _C, (2) incubation for RNA cleavage atroom temperature in a reaction buffer for a designated time, (3)addition of EDTA to 30 mM to stop the reaction, (4) separation ofcleavage products by denaturing 10% PAGE, and (5) quantitation using aPhospholmager and ImageQuant software. Aliquots of an RNA cleavagereaction solution were collected at different reaction time points thatwere all under 10% completion and the rate constant for the reaction wasdetermined by plotting the natural logarithm of the fraction of DNA thatremained unreacted vs. the reaction time. The negative slope of the lineproduced by a least-squares fit to the data was taken as the rateconstant.

Example 5 Selection Scheme for the Isolation of a DNA Enzyme

Since F1QDNA had the largest fluorescence intensity increase, an RNAlinkage immediately flanked by a fluorophore-containing nucleotide and aquencher-modified nucleotide was incorporated into the startingrandom-sequence pool to be used for the creation of DNA enzymes. Aselection scheme to isolate signaling autocatalytic DNA molecules isshown in FIG. 3. The general scheme is shown in FIG. 3A and the specificsequences of a preferred embodiment are shown in FIG. 3B. In step I, apool of single-stranded 86-nt DNA containing 43 random-sequencenucleotides is prepared. This is termed Library L1. In the sequenceshown in FIG. 3B, N₄₃ denotes the random sequence of 43 nucleotides. 300pmol of 5_-phosphorylated, gel-purified, 86-nt random-sequence DNA L1was mixed in an equimolar ratio with template T1 and acceptor A1 (allsequences shown in FIG. 3B), heated to 90° C. for 30 sec, cooled to roomtemperature, and combined with 10_ligase buffer and T4 DNA ligase forDNA ligation to introduce the modified DNA domain. (Step I, FIG. 3A) Theligation mixture (50 _l) contained 50 mM Tris-HCl (pH 7.8 at 23° C.), 40mM NaCl, 10 mM MgCl₂, 1 mg/ml BSA, 0.5 mM ATP, and 0.1 U (Weiss) _L⁻¹ T4DNA ligase. The solution was incubated at 23° C. for 1 hr and theligated 109-nt DNA was purified by 10% denaturing PAGE. (Step II)

The 109-nt DNA population constructed as above was used as the initialpool (denoted generation 0 or G0), which was heated to 90° C. for 30seconds, cooled to room temperature, and then combined with a2_selection buffer (100 mM HEPES, pH 6.8 at 23° C., 800 mM NaCl, 200 mMKCl, 15 mM MgCl₂, 10 mM MnCl₂, 2.5 mM CdCl₂, 2 mM CoCl₂, 0.5 mM NiCl₂)to a final DNA concentration of 0.05 _M. (Step III) The mixture wasincubated for self-cleavage at 23° C. for 5 hr.

The cleavage reaction was stopped by the addition of EDTA (pH 8.0) to afinal concentration of 30 mM. The cleaved DNA was isolated by 10%denaturing PAGE. To increase the yield of DNA recovery and to track thestatus of 94-nt cleaved product, 0.25 pmol of strongly radioactive 94-ntDNA marker made by alkaline digestion of the 109-nt construct was usedas the Acarrier DNA≅. The isolated cleavage product was amplified by PCRin 5_(—)100 _(—)1 reaction volume using primers P1 and P2 (FIG. 3B)(Step IV). The PCR reaction was monitored in real-time using SYBR Green(Molecular Probes). 2% of the amplified DNA product was used as the DNAtemplate for a new PCR reaction in a 10_(—)100 _(—)1 reaction volumeusing primer P1 and ribo-terminated primer P3 (Step V). The reactionmixture also included 30 _Ci of [_-³²P]dGTP for DNA labeling.

The DNA product in the second PCR was recovered by ethanolprecipitation, resuspended in 90 _L of 0.25 M NaOH and incubated at 90°C. for 10 min to cleave the single embedded RNA linkage. (Step VI) Thecleavage solution was neutralized by adding 10 _L of 3 M NaOAc (pH 5.2at 23° C.) and ˜86-nt single-stranded DNA fragment was isolated bydenaturing 10% PAGE. The recovered DNA molecules were incubated with 10units of PNK at 37° C. for 1 hr for DNA phosphorylation in a 100-_(—)1reaction mixture containing 50 mM Tris-HCl (pH 7.8 at 23° C.), 40 mMNaCl, 10 mM MgCl₂, 1 mg/ml BSA and 0.5 mM ATP. The reaction was stoppedby the addition of EDTA to a final concentration of 30 mM. The5¹-phosphorylated DNA was used for the second round of selection usingthe same procedure described for the first round of selection.

In this example, Mg²⁺ and several divalent transition metal ionsincluding Mn²⁺, Co²⁺, Ni²⁺ and Cd²⁺ were included in the selectionbuffer. The total concentration of divalent metal ions was chosen to be15 mM with individual concentrations set at the following: 7.5 mM Mg²⁺,5 mM Mn²⁺, 1.25 mM Cd²⁺, 1 mM Co²⁺, 0.25 mM Ni²⁺. It is clearly apparentthat other combinations and concentrations may also be effective.

Repeated rounds of selection lead to the selection of a highly efficientdeoxyribozyme. The selection progress is summarized in FIG. 3C. None orlow cleavage activity was observed for DNA sequences isolated ingenerations G0-G8. However, significant cleavage was seen in G9 and G10.By G11, more than 30% of the DNA construct was cleaved after a 5-hrincubation. The reaction time was then progressively reduced in order toderive very efficient DNA enzymes. The self-cleavage reaction wasallowed to proceed for only 10 minutes in G12 and 1 minute in G13, andthe reaction time was further reduced to 30 seconds in G14 and G15, to 5seconds in G16 and G17, and finally to about 1 second in G18-G21. TheDNA molecules in G22 were allowed to react for 1 minute and the cleavedDNA was cloned.

Example 6 Cloning and Sequencing of Selected Deoxyribozymes

DNA sequences from the 22nd round of selection were amplified by PCR andcloned into a vector by the TA cloning method. The plasmids containingindividual catalysts were prepared using a Qiagen MiniPrep Kit. DNAsequencing was performed on an LCQ2000 capillary DNA sequencer(Beckman-Coulter) following the procedures recommended by themanufacturer.

Example 7 Isolation and Activity of an Autocalytic DNA Molecule

A single class of deoxyribozyme was found in the G22 pool after morethan 20 clones were sequenced. The sequence of this autocatalytic DNAmolecule, named DEC22-18, is given in FIG. 4A. The confirmation of itscatalytic activity and the analysis of its metal ion requirements areshown FIG. 4B. The DNA enzyme was labeled at the phosphodiester bondlinking the 23^(rd) and 24^(th) nucleotides with ³²P. The uncleaved109-nt DEC22-18 is therefore weakly fluorescent (since the Q moiety isstill present) and highly radioactive. As shown in FIG. 4B uponself-cleavage, DEC22-18 gives rise to two cleavage products, with the5=cleaved fragment (15-nt; P1) being strongly fluorescent but notradioactive and the 3=fragment (94-nt; P2) being only radioactive. Thetwo cleavage products were obtained by the partial digestion of thedeoxyribozyme with NaOH and used as the control (lane 1). When thedeoxyribozyme was treated with water (lane 2), monovalent metal ions(lane 3), Cd²⁺ (lane 5) or Mg²⁺ (lane 8), no cleavage product wasproduced; when the DNA enzyme was treated with Co²⁺ (lane 4), Ni²⁺ (lane6) or Mn²⁺ (lane 7), it self-cleaved into the two expected DNA fragmentswith the matching signaling properties. In each case, the ratio offluorescence intensity of P1 over that of uncleaved DEC22-18 wassignificantly larger than the ratio of radioactivity for these species,signifying a fluorescence enhancement consistent with the coupledcatalysis-signaling mechanism. The data indicate that DEC22-18 is ametallo DNA enzyme capable of using Co(II), Ni(II) or Mn(II) as thedivalent metal cofactor. Further experiments suggested that Co(II) is apreferred metal cofactor for DEC22-18.

Example 8 Determination of an Optimal Sequence

The optimal sequence for activity was determined using nucleotidetruncation experiments. The truncation strategy is shown in FIG. 5.DEC22-18 exhibits a k_(obs) of 1.0 min⁻¹ under the selection bufferconditions (50 mM HEPES, pH 6.8 at 23° C., 400 mM NaCl, 100 mM KCl, 7.5mM MgCl₂, 5 mM MnCl₂, 1 mM CoCl₂, 0.25 mM NiCl₂, 1.25 mM CdCl₂).DEC22-18 is a large DNA molecule consisting of 109 nucleotides. Todetermine whether the DNA enzyme sequence could be minimized, a seriesof DNA molecules were synthesized with variable truncations from the3′-end. These truncated mutants were examined for catalytic activity andthe results are summarized in FIG. 5 (relative activities are shown withthat of the wild-type DEC22-18 taken as 100). In one embodiment, theresults indicate that the last 29 nucleotides of DEC22-18 can be deletedwithout significantly reducing the catalytic activity. In otherembodiments, some truncated mutants are more effective than thewild-type molecule. In a preferred embodiment, the truncation of thelast 26 nucleotides produced a 83-nt enzyme, denoted DEC22-18A, that hadsignificantly improved catalytic activity with a k_(obs) of 2.1 min⁻¹under the selection buffer conditions. DEC22-18A is an even moreeffective catalyst when present in a solution containing 50 mM HEPES (pH6.8 at 23° C.), 5 mM MgCl₂, 10 mM CoCl₂, without monovalent metal ions.In this case, the self-cleavage reaction was too fast to allow anaccurate measurement of the rate constant using conventional manualquenching methods (data not shown). The k_(obs) value can be estimatedto be near 10 min⁻¹ based on the observation that nearly 50% of theDEC22-18A was cleaved in 3 seconds.

Example 9 Design of a Transacting DNA Enzyme

A trans acting DNA enzyme is provided. A secondary structure forDEC22-18A predicted by the M-fold program(http://bioinfo.math.rpi.edu/˜mfold/dna) is shown in FIG. 6A. Thisstructure was used to successfully design a trans-acting DNA enzymesystem, DET22-18, by replacing the stem-1 and its loop existing inDEC22-18A with a stem made of eight base-pairs. This structure is shownin FIG. 6B. DET22-18 is a true DNA enzyme and a multiple-turnover DNAenzyme that cleaves substrate S1 according to Michaelis-Menten kinetics.FIG. 6C shows the data from a kinetic experiment where DET22-18 was usedat 5 nM while the concentration of S1 was varied between 100-2000 nM. Ak_(cat) of 7.2_(—)0.7 min⁻¹ and a K_(M) of 0.94_(—0.19) _M were derivedusing GraFit software. These data indicate that the 58-nt DET22-18 is avery efficient DNA enzyme.

Example 10 Signaling Properties of DET22-18

The signaling behavior of the DET22-18/S1 substrate system was monitoredin real time via fluorescence spectroscopy and the results are shown inFIG. 7. A less than optimal Co(II) concentration (1 mM rather than 10mM) was used to slow down the cleavage reaction so that the fluorescenceintensity changes could be monitored using the conventionalspectroscopic method as well as to minimize any fluorescence quenchingimposed by this metal ion. The signaling reaction was examined under twodifferent enzyme: substrate ratios: (1) DET22-18 (E) in 10-fold excessover S1 (FIG. 7A) and (2) S1 in 10-fold excess over DET22-18 (FIG. 7B).In both cases, the system had a constant fluorescence intensity (first10 minutes of the reaction) when S1 was incubated with metal ions alonewithout DET22-18. When the DNA enzyme was introduced, the fluorescenceintensity of both solutions increased sharply. In FIG. 7A (E:S=10:1),the fluorescence enhancement (F/F0; F0 was the initial intensity and Fwas the intensity at any given time) increased at such a rapid rate thatwithin 1 minute, 7.4-fold enhancement was observed (see inset graph). InFIG. 7B, the fluorescence enhancement increased at a reduced rate asexpected because the concentration of the DNA enzyme was 10-fold lessthan that of the substrate. There was a 3.3-fold enhancement in 1-minuteincubation (see inset graph), representing an initial turnover rate of2.1/min (based on the observation that a 16-fold enhancement wasobserved when the reaction was completed). These data indicate that thesignaling DNA enzyme can be used for signal generation under a broadrange of substrate concentrations.

Example 11 Creation of a Signaling Allosteric DNA Enzyme

The stem-loop feature in the structure of DEC22-18A is ideal for thedesign of allosteric deoxyribozymes. To determine whether DEC22-18Acould be easily designed into an allosteric DNA enzyme, an ATP aptamerwas conjugated to the DNA enzyme through a weakened stem-1. Thisstructure is shown in FIG. 8A. In the absence of ATP, the weak stem-1does not associate strongly and as a result, the catalytic activity ofthis construct is fairly weak. However when ATP is introduced, theaptamer domain forms a stable complex with ATP to promote the formationof the stem-1 and thereby significantly increases the cleavage activity.

The conjugated DNA molecule or Aaptazyme≅ was assessed for signalingproperties initially under the following reaction conditions: 50 mMHEPES (pH 6.8 at 23° C.), 14 mM MgCl₂, 1 mM CoCl₂, 23° C. The resultsare shown in FIG. 8A. The fluorescent intensity increased at a rate of˜0.04 fluorescence unit/min (f.u./min) when ATP was absent (first5-minute incubation). Upon introduction of ATP (prior to the datarecording at the 6th minute of the incubation), the signaling rateincreased to ˜0.16 f.u./minXa 4-fold enhancement in the catalytic rate.The system reached 80% of its maximal signaling capability in 3 minutesfollowing the addition of ATP. The nature of the RNA-cleavage-dependentfluorescence signaling was confirmed by PAGE analysis of the cleavageproducts using a ³²P-labeled DNA construct. An identical 4-foldactivation of RNA cleavage by ATP was observed in the PAGE experiment.

The data shown in FIG. 8B suggests that the RNA cleavage activity of theaptamer-deoxyribozyme construct was high in the absence of ATP. Thissuggests that the enzymatic domain alone can form a sufficiently stableand active structure to render the efficient catalysis. To determinewhether this ability of “self-folding” could be weakened at a highertemperature and a reduced Co²⁺ concentration, a series of experiments atelevated temperatures and decreased Co²⁺ concentrations were performed.FIG. 8C illustrates the results from a set of experiments conducted at37° C. and 0.25 mM Co²⁺ in the presence of 1 mM ATP (triangles) or 1 mMGTP (squares). Each reaction mixture was incubated in the absence of ATPor GTP for first 10 minutes, and ATP or GTP was introduced before datarecording at the 11th minute of the reaction. In the absence of ATP andwith or without GTP, the reaction proceeded at the same signaling rate(initial rate) of 9.5_(—)10⁻⁴ f.u./min. In the presence of ATP, thesignaling rate increased to 1.8_(—)10⁻² f.u./min, representing a nearly20-fold of activation by ATP. The data in FIG. 8C also indicate that thetarget reporting was ATP-specific as GTP did not produce any significantsignal enhancement. Signaling DNA enzymes with more responsiveallosteric activation and less reduction in catalytic rate can beobtained through in vitro selection using partially randomized DEC22-18sequences.

Those skilled in the art will readily recognize that modifications andequivalents of the specific embodiments disclosed herein can be achievedusing no more than routine experimentation. Such modifications andequivalents are intended to be encompassed by the following claims.

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1. A signaling DNA construct comprising: i. an enzymatic DNA sequence; and ii. a DNA chain having a ribonucleotide linkage flanked by a fluorophore modified oligonucleotide and a quencher modified oligonucleotide in sufficient proximity to each other whereby, in the absence of catalysis, fluorescence from the fluorophore is quenched by the quencher.
 2. The signaling enzyme construct of claim 1 wherein the enzymatic DNA sequence is a cis-acting enzyme having the sequence defined in SEQ.ID.NO.7 or SEQ.ID.NO.8.
 3. The signaling DNA construct of claim 1, wherein the enzymatic DNA sequence is a trans-acting DNA enzyme having the sequence of SEQ.ID.NO.
 9. 4. The signaling DNA construct of claim 1, further comprising an aptamer sequence conjugated to the enzymatic DNA sequence.
 5. The signaling DNA construct of claim 4 comprising the sequence of SEQ.ID.NO
 10. 6. A method for the selection of an enzymatic DNA sequence, said method comprising inserting a random sequence into a DNA chain having a ribonucleotide linkage flanked by a fluorophore modified oligonucleotide and a quencher modified oligonucleotide and determining whether a fluorescent signal is generated wherein the fluorophore modified oligonucleotide and the quencher modified oligonucleotideare in sufficient proximity to each other whereby, in the absence of catalysis, fluorescence from the fluorophore is quenched by the quencher.
 7. A method for the detection of an enzymatic DNA sequence, said method comprising the steps of: i. providing a library of oligonucleotides to be screened; ii. ligating the oligonucleotides to an acceptor sequence comprising a ribonucleotide linkage flanked by a fluorophore modified oligonucleotide and a quencher modified oligonucleotide; and iii. determining whether a fluorescent signal is generated due to cleavage at the ribonucleotide linkage.
 8. A method for the selection of a DNA enzyme, said method comprising: i. providing a library of oligonucleotides to be screened; ii. ligating the oligonucleotides to an acceptor sequence comprising a ribonucleotide linkage flanked by a fluorophore modified oligonucleotide and a quencher modified oligonucleotide; iii. determining whether a fluorescent signal is generated due to cleavage at the ribonucleotide linkage; and iv. amplifying sequences which generate a fluorescent signal.
 9. A method for the selection of an aptamer sequence, said method comprising: i. conjugating a library of oligonucleotide sequences to a signaling DNA construct as defined in claim 1 to provide a conjugated molecule; ii. Incubating said conjugated molecule in the presence of a desired target; iii. determining whether a fluorescent signal is generated; and iv. amplifying sequences which generate a signal.
 10. A kit for the selection of an enzymatic DNA sequence, said kit comprising a DNA chain having a) a site for insertion of test nucleotide sequence; and b) a ribonucleotide linkage flanked by a fluorophore modified oligonucleotide and a quencher modified oligonucleotide in sufficient proximity to each other whereby, in the absence of catalysis, fluorescence from the fluorophore is quenched by the quencher and in the presence of a catalytic test nucleotide sequence, a fluorescent signal is generated.
 11. A method for the detection of a co-factor, said method comprising the steps of: i. providing a signaling DNA construct as defined in claim 1; ii. introducing a sample; and iii. determining whether a signal is generated, wherein, in the presence of a required co-factor, cleavage occurs at the ribonucleotide linkage and a fluorescent signal is generated.
 12. A method for the detection of a co-factor, said method comprising the steps of: i. providing a signaling DNA construct as defined in claim 4; ii. introducing a sample; and iii. determining whether a signal is generated, wherein, in the presence of a required co-factor, cleavage occurs at the ribonucleotide linkage and a fluorescent signal is generated. 